DNA repair pathways maintain the integrity of the genome and help prevent the onset of cancer. Conversely, DNA repair deficiencies engender tumor heterogeneity and promote the selection of highly aggressive subtypes (i.e., `mutator' phenotype). Importantly, DNA repair proteins have emerged as potential synthetic lethal targets for improving the selective response to currently available anti-cancer regimens. Key to advancing such a strategy is a detailed mechanistic understanding of the select DNA repair pathway in question, an appreciation of repair pathway redundancy & crosstalk and a molecular characterization of processes that regulate the function and stability of the proteins essential for repair. This completely revised R01 proposal builds on our recent discovery that Pol is ubiquitylated on lysines 206/244 in a manner that is dependent on the cell cycle and the type of DNA damage. Further, we reported that (i) Pol ubiquitylation may govern pathway crosstalk, (ii) select oxidized DNA lesions require/promote Pol degradation and (iii) Pol-mediated BER facilitates DNA repair pathway crosstalk that may be regulated by steady-state levels of Pol protein. Therefore, our first task was to identify the E3 ligases and de-ubiquitylation enzymes (DUBs) that may regulate Pol. High-resolution mass spectrometry proteomic analysis of Pol-interacting proteins and in response to PARP-activation allowed us to identify several E3 ligases and DUBs likely to target Pol to regulate stability and function. Our new data supports a role for the E3 ubiquitin ligase TRIP12 contributing to the regulation of Pol stability and governing Pol chromatin retention. Further, Pol ubiquitylation appears to govern BER repair complex dynamics. Most importantly, our preliminary data suggests that ubiquitylation facilitates trafficking of Pol to participate in late-phase replication associated repair (RAR) foci in response to complex DNA lesions. These RAR foci are XRCC1 dependent but devoid of DSB markers such as ?H2AX or 53BP1. The Aims detailed in the proposal will use purified proteins, cancer cell lines, tumor stem cells, high-resolution proteomics, live-cell fluorescent imaging and mouse xenografts to address our hypothesis that ubiquitylation/de-ubiquitylation regulates Pol stability, Pol-dependent BER repair complex dynamics and facilitates a coordinated trafficking mechanism to promote Pol involvement yet suppress 53BP1 involvement at complex lesions following alkylation, cisplatin and radiation damage.